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  • Title: Interactions between the amino- and carboxyl-terminal regions of G alpha subunits: analysis of mutated G alpha o/G alpha i2 chimeras.
    Author: Denker BM, Boutin PM, Neer EJ.
    Journal: Biochemistry; 1995 Apr 25; 34(16):5544-53. PubMed ID: 7727415.
    Abstract:
    Receptors activate the G alpha subunits of heterotrimeric G proteins by binding to the C-terminus and reducing their affinity for bound GDP, therefore promoting exchange of GDP for GTP. Although this general mechanism is the same for all G alpha subunits, different G alpha subunits vary in nucleotide binding and hydrolysis even though the residues that make up the guanine nucleotide binding site are virtually identical. We have shown previously that truncation of 14 amino acids from the C-terminus of G alpha o decreased the apparent affinity for GDP and permitted us to see an activated conformation with GTP [Denker, B. M., et al. (1992) J. Biol. Chem. 267, 9998-10002]. To test whether mutations in the receptor binding region lead to different phenotypes in closely related G alpha subunits, we made the equivalent deletions in G alpha i2, synthesized the proteins in vitro in a rabbit reticulocyte lysate and used the pattern of native tryptic proteolysis as an index of conformation. The phenotype of truncated G alpha i2 was different from that of truncated G alpha o: GDP affinity was reduced, but we could not detect an activated conformation with GTP (although GTP gamma S activated normally). Analysis of shorter deletions showed that loss of three hydrophobic residues (between 11 and 13 residues from the C-terminus) was responsible for the phenotypes. To define the regions of G alpha o and G alpha i2 that were responsible for their different phenotypes, we used a conserved BamHI site (codon 212) to make chimeras. Each chimera truncated at the C-terminus had the phenotype of the donor of the amino-terminal portion. Both truncated chimeras were activated by GTP gamma S-like wild-type proteins, and both had decreased apparent affinity for GDP. Full-length chimeric subunits behaved like wild-type proteins. The crystal structure of G alpha t and G alpha i1 shows that the three hydrophobic amino acids we have identified make contact with residues in the N- and C-terminal portions of the protein. Our studies point to the importance of the contacts in the N-terminal region (start of beta strands 1 and 3) that may stabilize the C-terminal alpha helix, affect nucleotide binding, and determine the characteristic features of different G alpha subunits.
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